Mutations and deletions in the mitochondrial genome are linked to a spectrum of diseases, but the concept that mitochondrial (mt) DNA is a target of reactive species in acute, free radical-mediated pulmonary disorders has not been considered. Because of their pathological relevance, we used rat pulmonary artery (PA), pulmonary microvascular (MV), and pulmonary vein (PV) ECs in experiments to explore the relation between xanthine oxidase (XO)-induced mtDNA damage and loss of cell viability. The rank order of XO-induced mtDNA damage was the same as the rank order of XO-mediated cytotoxicity. Interestingly, survival after XO treatment was inversely related to mtDNA repair capacity; the most resistant phenotype, MVECs, repaired XO-induced mtDNA damage rapidly, followed by PA and PVECs. The importance of mtDNA was further suggested by the finding that transfection of PAECs with the mtDNA repair enzyme, ogg-1, reduced XO-induced mtDNA damage and attendant cytotoxicity. Finally, we determined that the fragile mtDNA sequence incriminated in human diseases was prone to XO- mediated damage in PAECs. Based on these considerations, we proposed to test the working hypothesis that mtDNA serves as a sentinel molecule in which excessive or persistent damage to specific sequences causes pulmonary vascular EC death due to activation of mitochondrial pathway of apoptosis. Using rat pulmonary vascular ECs as model systems, we will test the hypothesis that: (1) The capacity to repair different patterns of mtDNA lesions is predictive of EC phenotype sensitivity to reactive species-mediated damage to the mitochondrial genome and to apoptotic cell death; (2) Increased mtDNA repair capacity protects against reactive species-induced mtDNA damage; mitochondrial dysfunction, and activation of the mitochondrial pathway of apoptosis; and (3) Functionally-significant sequences within the mtDNA "heavy strand" are prone to reactive species-induced damage and damage to these sequences is linked to depressed mitochondrial gene expression and ATP content. The proposed research will provide new insight into factors governing EC responses to free radicals. If lung EC phenotypes exhibit divergent sensitivities to free radical stress, this will have critical implications for understanding the cellular basis of acute lung injury.